Rifamycins are effective inhibitors of a wide range of bacterial RNA polymerases, earning their place as first line antibiotics in treatment of tuberculosis. However, their clinical efficiency and versatility are severely limited by the rapid rise of resistant strains. The majority of the rifamycin-resistant mutations map to the rpoB gene, encoding the subunit of RNA polymerase. Since this enzyme is both the target of and the source of resistance to rifamycins, elucidation of its interactions with rifamycins is instrumental for design of new, more potent antibiotics and expanding their use to new therapeutic targets. The structure of the rifampicin-polymerase complex has been obtained, and the mechanism of inhibition has been proposed. However, the current model fails to account for many aspects of rifamycin action, e.g., the existence ofrpoB mutants that are resistant to rifampicin yet sensitive to its derivatives, ruling out the simple loss-of-binding mechanism. A combination of genetic and biochemical approaches will be used to determine the molecular mechanism of rifamycin-resistance. First, a comprehensive mutagenesis of the E. coli rpoB gene will be used to isolate mutants resistant to different rifamycins (rifamycin SV, rifampin, rifabutin, etc), with the emphasis on the detailed analysis of mutants conferring differential resistance as the key to understanding rifamycin action. Second, RNA polymerase variants corresponding to these mutants will be purified to confirm the antibiotic resistance in a highly purified system in vitro; effective changes in transcription kinetics and thermodynamics will be determined. Third, using radio labeled rifamycin, physical parameters of rifamycins binding to different states of RNA polymerase will be quantified. Finally, single nucleotide resolved transcription assays will be used to determine the effects of various chemical modifications to basic rifamycin pharmacophore, especially those present in clinically important antibiotics rifampin and rifabutin. The data obtained as a result of this systematic analysis will be used to refine the structural model of rifamycin-RNA polymerase complex and to develop explicit kinetic and thermodynamic models of rifamycin action. Finally a set of rules for design of more potent rifamycin-like antibiotics will be formulated to achieve the long-term goal of knowledge-based creation of the new generation of antibacterial drugs.